Design and development of nano immuno-sensor for ultra-sensitive detection of gastrin-glycine using recombinant antibody fragments

Abstract

Gastric cancer (GC) is one of the major health problems. However, this lethal problem can be amended by early detection. The novel technology of biomarkers can offer valuable information about the status of malignancy. Gastrin extended glycine (G17-Gly) is a product of gastrin gene, which act as the enhancer of the proliferation of cancerous cells and preventing anticancer immune reactions. In this research, a novel and sensitive immunosensor for electrochemical determination of G17-Gly was designed based on signal amplification and tailor-made recombinant antibody technology. G17-Gly-antibody fragment (anti-G17-Gly scFv) was immobilized onto an innovative nanocomposite comprising of poly Cetyl Trimethyl Ammonium Bromide (P (CTAB) as conductive matrix, chitosan (CS) as biocompatible agent and gold nanoparticles (AuNPs) as signal amplification element. The small size of scFv and VL and their low risk of triggering antibody response turn it to the finest alternative for conventional whole antibodies in therapeutic, analytic and research applications. Additionally, exploiting the whole phage, which displays the antibody fragments on its body, as the biorecognition element further enhances the effieciency of the proposed immunosensors. The benefits of AuNPs such as, large specific surface area and exceptional biocompatibility, laid the foundation for the successful covalent binding between antibody and AuNPs for the construction of immunosensor. High surface area provided by AuNPs and the small size of scFv establish the basis for immobilizing a high amount of the anti-G17-Gly on the surface of electrode for detecting G17-Gly in human plasma samples. Materials and methods Fabrication of the immunosensor was based on the electrodeposition of P(CTAB-CS)-AuNPs on gold electrodes and incubation of biological materials on the surface of modified GE. The utilized matrix, specified antibody fragments, and whole phages, besides increasing the electrodes available surface area, provide a proper electroactive site, large number of immobilized bioreceptors and their appropriate orientation for binding to the analyte. Results Under optimal conditions, designed immunosensors provide an excellent analytical capability for detecting and determining G17-Gly in human plasma with a linear range from 0.5 mM-0.05 pM and LLOQ of 0.05 pM. Furthermore, satisfactory results are obtained for the determination of G17-Gly in human plasma samples, indicating the potential of the manufactured immunosensors to be applied in clinical analysis. Conclusion In summary, the P(CTAB-CS)-AuNPs/Ab/BSA/Ag immunosensors were successfully fabricated to detect G17-Gly biomarker in human plasma samples. The use of antibody fragments instead of whole antibodies represented outstanding benefits such as, small size, customizability, easy production, high selectivity, and high amount of immobilized biorecognition elements. In addition, utilizing the whole phage displaying scFv and VL as the biorecognition element, further enhanced both the number of recognition sites and their orientation. As a result the sensitivity and selectivity of the designed immunosensors boosted up considerably. The aforementioned characteristics demonstrate that the projected immunosensors can be used in biological and clinical diagnosis as reliable tools for identifying different oncobiomarkers.